Non-human animals having a humanized signal-regulatory protein gene

ABSTRACT

Genetically modified non-human animals and methods and compositions for making and using the same are provided, wherein the genetic modification comprises a humanization of an endogenous signal-regulatory protein gene, in particular a humanization of a SIRPα gene. Genetically modified mice are described, including mice that express a human or humanized SIRPα protein from an endogenous SIRPα locus.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/866,632, filed Jan. 10, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/615,298, filed Jun. 6, 2017, now U.S. Pat. No.9,901,083, which is a continuation of U.S. patent application Ser. No.15/263,916, filed Sep. 13, 2016, now U.S. Pat. No. 9,700,027, which is acontinuation of U.S. patent application Ser. No. 14/882,531, filed Oct.14, 2015, now U.S. Pat. No. 9,462,794, which is a continuation of U.S.patent application Ser. No. 14/493,745, filed Sep. 23, 2014, now U.S.Pat. No. 9,193,977, which claims the benefit of priority of U.S.Provisional Application No. 61/881,261, filed Sep. 23, 2013, the entirecontents of which are incorporated herein by reference.

BACKGROUND

The immune system is composed of several different cell types that areinvolved in multiple highly regulated processes and together generateimmune responses that are effective in eliminating foreign proteins.Further, these same immune cells have been found to possess aself-awareness property by virtue of, inter alia, regulatory membraneproteins that regulate cell-to-cell interactions. Such communication iscritical for the survival of such organisms, as these same proteins aresuggested to be an important determinant of transplant engraftment.However, no in vivo system exists to determine the molecular aspects ofhuman immune cell-to-cell interactions and its regulation. Such a systemprovides a source for assays in human hematopoietic and immune systemrelated functions in vivo, identification of novel therapies andvaccines.

SUMMARY OF INVENTION

The present invention encompasses the recognition that it is desirableto engineer non-human animals to permit improved engraftment of humanhematopoietic stem cells. The present invention also encompasses therecognition that non-human animals having a humanized SIRPα gene and/orotherwise expressing, containing, or producing a human or humanizedSIRPα protein are desirable, for example for use in engraftment of humanhematopoietic stem cells.

In some embodiments, a non-human animal of the present inventionexpresses a SIRPα polypeptide comprising an extracellular portion of ahuman SIRPα protein and intracellular portion of a mouse SIRPα protein.

In some embodiments, an extracellular portion of a human SIRPα proteincomprises amino acids corresponding to residues 28-362 of a human SIRPαprotein that appears in SEQ ID NO: 4.

In some embodiments, an extracellular portion of a human SIRPα proteinshares a percent identity of at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% with a correspondingextracellular portion of a human SIRPα protein that appears in Table 3.In some embodiments, an extracellular portion of a human SIRPα proteinshares 100% identity (or identical) with a corresponding extracellularportion of a human SIRPα protein that appears in Table 3.

In some embodiments, a non-human animal of the present invention doesnot also express an endogenous non-human SIRPα protein. In someembodiments, the non-human animal is a rodent and does not also expressan endogenous rodent SIRPα protein. In some embodiments, the non-humananimal is a mouse and does not also express an endogenous mouse SIRPαprotein having a sequence that appears in Table 3.

In some embodiments, the present invention provides a non-human animalcomprising a SIRPα gene that comprises exons 2, 3 and 4 of a human SIRPαgene operably linked to a non-human SIRPα promoter.

In some embodiments, a SIRPα gene of a non-human animal of the presentinvention comprises exons 1, 5, 6, 7 and 8 of an endogenous non-humanSIRPα gene.

In various embodiments, a non-human animal of the present invention is arodent. In some certain embodiments, a rodent of the present inventionis selected from a mouse or a rat.

In some embodiments, the present invention provides a SIRPα polypeptideencoded by the gene of a non-human animal as described herein.

In some embodiments, the present invention provides a cell or tissueisolated from a non-human animal as described herein. In someembodiments, a cell is selected from a lymphocyte (e.g., a B or T cell),a myeloid cell (e.g., a macrophage, a neutrophil, a granulocyte, amyeloid dendritic cell, and a mast cell), and a neuron. In someembodiments, a tissue is selected from adipose, bladder, brain, breast,bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node,muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis,ovum, and/or a combination thereof.

In some embodiments, the present invention provides an isolated mousecell or tissue whose genome includes a SIRPα gene that encodes theextracellular portion of a human SIRPα protein linked to theintracellular portion of a mouse SIRPα protein. In some embodiments, aSIRPα gene of the present invention is operably linked to a mouse SIRPαpromoter. In some embodiments, a SIRPα gene of the present inventioncomprises exons 2, 3, and 4 of a human SIRPα gene.

In some embodiments, the present invention provides a non-humanembryonic stem (ES) cell whose genome comprises a SIRPα gene asdescribed herein. In some embodiments, the ES cell comprises exons 2, 3and 4 of a human SIRPα gene operably linked to a non-human SIRPαpromoter. In some certain embodiments, the ES cell is a rodent ES cell.In some embodiments, a non-human embryonic stem cell of the presentinvention is a mouse or rat embryonic stem cell.

In some embodiments, the present invention provides a non-human embryocomprising, made from, obtained from, or generated from a non-humanembryonic stem cell comprising a SIRPα gene as described herein. In someembodiments, a non-human embryo of the present invention is a rodentembryo. In some embodiments, a rodent embryo as described herein is amouse or rat embryo.

In some embodiments, the present invention provides a method of making anon-human animal that expresses a SIRPα protein from an endogenous SIRPαlocus, wherein the SIRPα protein comprises a human sequence, the methodcomprising targeting an endogenous SIRPα locus in a non-human ES cellwith a genomic fragment comprising a nucleotide sequence that encodes ahuman SIRPα protein in whole or in part; obtaining a modified non-humanES cell comprising an endogenous SIRPα locus that comprises said humansequence, and, creating a non-human animal using said modified ES cell.

In some embodiments, said nucleotide sequence comprises exons 2, 3 and 4of a human SIRPα gene. In some embodiments, said nucleotide sequencecomprises exons 2, 3 and 4 of a human SIRPα gene having a sequence atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80% N, at least 85%, at least 90%, at least 95%, orat least 98% identical to a human SIRPα gene that appears in Table 3.

In some embodiments, said nucleotide sequence encodes amino acidresidues 28-362 of a human SIRPα protein. In some embodiments, saidnucleotide sequence encodes amino acid residues 28-362 of a human SIRPαprotein having a sequence at least 50%, at least 55%, at least 60%, atleast 65%, at least 70% N, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to a human SIRPαprotein that appears in Table 3.

In some embodiments, the present invention provides a method ofproviding a mouse whose genome includes a SIRPα gene that encodes theextracellular portion of a human SIRPα protein linked to theintracellular portion of a mouse SIRPα protein, the method comprisingmodifying the genome of a mouse so that it comprises a SIRPα gene thatencodes the extracellular portion of a human SIRPα protein linked to theintracellular portion of a mouse SIRPα protein thereby providing saidmouse. In some embodiments, the SIRPα gene is a SIRPα gene as describedherein. In some embodiments, the SIRPα gene comprises exons 2, 3, and 4of a human SIRPα gene.

In some embodiments, the present invention provides a method ofengrafting human cells into a mouse, the method comprising steps ofproviding a mouse whose genome comprises a SIRPα gene that encodes theextracellular portion of a human SIRPα protein linked to theintracellular portion of a mouse SIRPα protein, and transplanting one ormore human cells into the mouse. In some certain embodiments, the methodfurther comprises as step assaying engraftment of the one or more humancells in the mouse. In some certain embodiments, the step of assayingcomprises comparing the engraftment of the one or more human cells tothe engraftment in one or more wild-type mice. In some certainembodiments, the step of assaying comprises comparing the engraftment ofthe one or more human cells to the engraftment in one or more mice whosegenome does not comprise a SIRPα gene that encodes the extracellularportion of a human SIRPα protein linked to the intracellular portion ofa mouse SIRPα protein.

In some embodiments, the human cells are hematopoietic stem cells. Insome embodiments, the human cells are transplanted intravenously. Insome embodiments, the human cells are transplanted intraperitoneally. Insome embodiments, the human cells are transplanted subcutaneously.

In some embodiments, the present invention provides a method comprisingthe steps of providing one or more cells whose genome includes a SIRPαgene that encodes the extracellular portion of a human SIRPα proteinlinked to the intracellular portion of a mouse SIRPα protein, incubatingthe one or more cells with a labeled substrate, and measuringphagocytosis of the labeled substrate by the one or more cells. In someembodiments, the cells are mouse cells.

In some embodiments, the substrate is fluorescently labeled. In someembodiments, the substrate is labeled with an antibody. In someembodiments, the substrate is one or more red blood cells. In someembodiments, the substrate is one or more bacterial cells.

In some embodiments, the present invention provides a method comprisingthe steps of providing a mouse whose genome includes a SIRPα gene thatencodes the extracellular portion of a human SIRPα protein linked to theintracellular portion of a mouse SIRPα protein, exposing the mouse to anantigen, and measuring phagocytosis of the antigen by one or more cellsof the mouse. In some embodiments, the step of exposing comprisesexposing the mouse to an antigen that is fluorescently labeled. In someembodiments, the step of exposing comprises exposing the mouse to one ormore cells that comprise the antigen. In some embodiments, the step ofexposing comprises exposing the mouse to one or more human cellscomprising the antigen. In some embodiments, the step of exposingcomprises exposing the mouse to one or more bacterial cells comprisingthe antigen.

In various embodiments, a SIRPα gene of the present invention comprisesexons 2, 3, and 4 of a human SIRPα gene. In various embodiments, anextracellular portion of a human SIRPα protein of the present inventioncomprises amino acids corresponding to residues 28-362 of a human SIRPαprotein that appears in Table 3. In various embodiments, a SIRPα gene ofthe present invention is operably linked to a mouse SIRPα promoter.

In some embodiments, the present invention provides a non-human animalobtainable by methods as described herein. In some certain embodiments,non-human animals of the present invention do not detectably express anextracellular portion of an endogenous SIRPα protein.

In some embodiments, the present invention provides methods foridentification or validation of a drug or vaccine, the method comprisingthe steps of delivering a drug or vaccine to a non-human animal asdescribed herein, and monitoring one or more of the immune response tothe drug or vaccine, the safety profile of the drug or vaccine, or theeffect on a disease or condition. In some embodiments, monitoring thesafety profile includes determining if the non-human animal exhibits aside effect or adverse reaction as a result of delivering the drug orvaccine. In some embodiments, a side effect or adverse reaction isselected from morbidity, mortality, alteration in body weight,alteration of the level of one or more enzymes (e.g., liver), alterationin the weight of one or more organs, loss of function (e.g., sensory,motor, organ, etc.), increased susceptibility to one or more diseases,alterations to the genome of the non-human animal, increase or decreasein food consumption and complications of one or more diseases.

In some embodiments, the present invention provides use of a non-humananimal of the present invention in the development of a drug or vaccinefor use in medicine, such as use as a medicament.

In some embodiments, the present invention provides use of a non-humananimal described herein to assess the efficacy of a therapeutic drugtargeting human cells. In various embodiments, a non-human animal of thepresent invention is transplanted with human cells, and a drug candidatetargeting such human cells is administered to the animal. The efficacyof the drug is determined by monitoring the human cells in the non-humananimal after the administration of the drug.

In various embodiments, non-human animals of the present invention arerodents, preferably a mouse or a rat.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The drawing included herein is for illustration purposes only not forlimitation.

FIG. 1 shows a diagram, not to scale, of an endogenous murine SIRPα gene(top) with each exon numbered. A humanized endogenous SIRPα gene(bottom) is shown containing exons 2-4 of a human SIRPα gene and aneomycin selection cassette (Ub-Neo) flanked by site-specificrecombinase recognition sites (e.g., loxP). The targeted insertion ofexons 2-4 of a human SIRPα gene results in an endogenous gene thatexpresses a humanized SIRPα gene having an extracellular regioncorresponding to a human SIRPα protein.

FIG. 2 shows an overlay of SIRPα expression of wild type and miceheterozygous for a humanized SIRPα gene.

FIG. 3 shows the percent of CD45⁺ cells in different strains of miceengrafted with human CD34+ cells.

FIG. 4 shows the percent of CD45⁺CD3⁺ cells in different strains of miceengrafted with human CD34 cells.

FIG. 5 shows the percent of CD45⁺CD19⁺ cells in different strains ofmice engrafted with human CD34⁺ cells.

FIG. 6 shows that Ab 1 suppressed growth of Raji tumors in adose-dependent manner in hCD34+ engrafted SIRPα BRG mice. Raji tumorvolume was measured on days 3, 6, 9, 13, 16, 20, 23, 27, 30 and 34 posttumor implantation. Data for individual animals (Panels A-D) ispresented. hCD34+ engrafted SIRPα BRG mice were administered 2×10⁶ Rajitumor cells subcutaneously on Day 0. Control groups received no antibody(vehicle control) (Panel A). For experimental groups, on Day 0 mice weretreated with an IP dose of a non-binding control Ab (control Ab 5) at0.4 mg/kg (Panel B), or Ab 1 at 0.4 mg/kg (Panel C) or 0.04 mg/kg (PanelD), followed by twice weekly doses for the length of the study. Thecomposite data for all individual test groups are shown in FIG. 7.

FIG. 7 shows that Ab 1 significantly suppressed growth of Raji tumorscompared to controls in hCD34+ engrafted SIRPα BRG mice. Data representsthe composite data from n=4-5 mice per group as shown in FIG. 6. Dataare expressed as mean (SEM) and were analyzed using analysis of variance(ANOVA) and post hoc tests to probe significant effects (Tukey's fortwo-way ANOVA). One mouse in the vehicle control group, Control Ab 5group, and Ab 1 0.4 mg/kg group was excluded from this composite graphdue to early death in order to analyze data by two-way ANOVA.

FIG. 8 shows that Ab 1 did not affect body weight in hCD34+ engraftedSIRPα BRG mice. Body weights were measured on days 3, 6, 9, 13, 16, 20,23, 27, 30 and 34 post tumor implantation. Data for individual animals(Panels A-D) was measured. hCD34+ engrafted SIRPα BRG mice wereadministered 2×10⁶ Raji tumor cells subcutaneously on Day 0. Controlgroups received no antibody (vehicle control) (Panel A). Forexperimental groups, on Day 0 mice were treated with an IP dose of theIgG1 non-binding Control Ab 5 at 0.4 mg/kg (Panel B) or Ab 1 at 0.4mg/kg (Panel C) or 0.04 mg/kg (Panel D), followed by twice weekly dosesfor the length of the study.

DEFINITIONS

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference.

The term “approximately” as applied herein to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain embodiments, the term “approximately” or “about” refers to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 90%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less ineither direction (greater than or less than) of the stated referencevalue unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

The term “biologically active” as used herein refers to a characteristicof any agent that has activity in a biological system, in vitro or invivo (e.g., in an organism). For instance, an agent that, when presentin an organism, has a biological effect within that organism, isconsidered to be biologically active. In particular embodiments, where aprotein or polypeptide is biologically active, a portion of that proteinor polypeptide that shares at least one biological activity of theprotein or polypeptide is typically referred to as a “biologicallyactive” portion.

The term “comparable”, as used herein, refers to two or more agents,entities, situations, sets of conditions, etc. that may not be identicalto one another but that are sufficiently similar to permit comparisonthere between so that conclusions may reasonably be drawn based ondifferences or similarities observed. Those of ordinary skill in the artwill understand, in context, what degree of identity is required in anygiven circumstance for two or more such agents, entities, situations,sets of conditions, etc. to be considered comparable.

The term “conservative” as used herein to describe a conservative aminoacid substitution refers to substitution of an amino acid residue byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of interest of a protein, for example, the abilityof a receptor to bind to a ligand. Examples of groups of amino acidsthat have side chains with similar chemical properties include aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45, hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

The term “disruption” as used herein refers to the result of ahomologous recombination event with a DNA molecule (e.g., with anendogenous homologous sequence such as a gene or gene locus. In someembodiments, a disruption may achieve or represent an insertion,deletion, substitution, replacement, missense mutation, or a frame-shiftof a DNA sequence(s), or any combination thereof. Insertions may includethe insertion of entire genes or fragments of genes, e.g. exons, whichmay be of an origin other than the endogenous sequence. In someembodiments, a disruption may increase expression and/or activity of agene or gene product (e.g., of a protein encoded by a gene). In someembodiments, a disruption may decrease expression and/or activity of agene or gene product. In some embodiments, a disruption may altersequence of a gene or an encoded gene product (e.g., an encodedprotein). In some embodiments, a disruption may truncate or fragment agene or an encoded gene product (e.g., an encoded protein). In someembodiments, a disruption may extend a gene or an encoded gene product;in some such embodiments, a disruption may achieve assembly of a fusionprotein. In some embodiments, a disruption may affect level but notactivity of a gene or gene product. In some embodiments, a disruptionmay affect activity but not level of a gene or gene product. In someembodiments, a disruption may have no significant effect on level of agene or gene product. In some embodiments, a disruption may have nosignificant effect on activity of a gene or gene product. In someembodiments, a disruption may have no significant effect on either levelor activity of a gene or gene product.

The phrase “endogenous locus” or “endogenous gene” as used herein refersto a genetic locus found in a parent or reference organism prior tointroduction of a disruption, deletion, replacement, alteration, ormodification as described herein. In some embodiments, the endogenouslocus has a sequence found in nature. In some embodiments, theendogenous locus is wild type. In some embodiments, the referenceorganism is a wild-type organism. In some embodiments, the referenceorganism is an engineered organism. In some embodiments, the referenceorganism is a laboratory-bred organism (whether wild-type orengineered).

The phrase “endogenous promoter” refers to a promoter that is naturallyassociated, e.g., in a wild-type organism, with an endogenous gene.

The term “heterologous” as used herein refers to an agent or entity froma different source. For example, when used in reference to apolypeptide, gene, or gene product or present in a particular cell ororganism, the term clarifies that the relevant polypeptide, gene, orgene product 1) was engineered by the hand of man; 2) was introducedinto the cell or organism (or a precursor thereof) through the hand ofman (e.g., via genetic engineering); and/or 3) is not naturally producedby or present in the relevant cell or organism (e.g., the relevant celltype or organism type).

The term “host cell”, as used herein, refers to a cell into which aheterologous (e.g., exogenous) nucleic acid or protein has beenintroduced. Persons of skill upon reading this disclosure willunderstand that such terms refer not only to the particular subjectcell, but also is used to refer to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. In some embodiments, a host cell isor comprises a prokaryotic or eukaryotic cell. In general, a host cellis any cell that is suitable for receiving and/or producing aheterologous nucleic acid or protein, regardless of the Kingdom of lifeto which the cell is designated. Exemplary cells include those ofprokaryotes and eukaryotes (single-cell or multiple-cell), bacterialcells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp.,etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S.cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells,insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells,Trichoplusia ni, etc.), non-human animal cells, human cells, or cellfusions such as, for example, hybridomas or quadromas. In someembodiments, the cell is a human, monkey, ape, hamster, rat, or mousecell. In some embodiments, the cell is eukaryotic and is selected fromthe following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS(e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA,MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065,HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3,L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell,HT1080 cell, myeloma cell, tumor cell, and a cell line derived from anaforementioned cell. In some embodiments, the cell comprises one or moreviral genes, e.g., a retinal cell that expresses a viral gene (e.g., aPER.C6™ cell). In some embodiments, a host cell is or comprises anisolated cell. In some embodiments, a host cell is part of a tissue. Insome embodiments, a host cell is part of an organism.

The term “humanized”, is used herein in accordance with itsart-understood meaning to refer to nucleic acids or proteins whosestructures (i.e., nucleotide or amino acid sequences) include portionsthat correspond substantially or identically with structures of aparticular gene or protein found in nature in a non-human animal, andalso include portions that differ from that found in the relevantparticular non-human gene or protein and instead correspond more closelywith comparable structures found in a corresponding human gene orprotein. In some embodiments, a “humanized” gene is one that encodes apolypeptide having substantially the amino acid sequence as that of ahuman polypeptide (e.g., a human protein or portion thereof—e.g.,characteristic portion thereof). To give but one example, in the case ofa membrane receptor, a “humanized” gene may encode a polypeptide havingan extracellular portion having an amino acid sequence as that of ahuman extracellular portion and the remaining sequence as that of anon-human (e.g., mouse) polypeptide. In some embodiments, a humanizedgene comprises at least a portion of an DNA sequence of a human gene. Insome embodiment, a humanized gene comprises an entire DNA sequence of ahuman gene. In some embodiments, a humanized protein comprises asequence having a portion that appears in a human protein. In someembodiments, a humanized protein comprises an entire sequence of a humanprotein and is expressed from an endogenous locus of a non-human animalthat corresponds to the homolog or ortholog of the human gene.

The term “identity” as used herein in connection with a comparison ofsequences, refers to identity as determined by a number of differentalgorithms known in the art that can be used to measure nucleotideand/or amino acid sequence identity. In some embodiments, identities asdescribed herein are determined using a ClustalW v. 1.83 (slow)alignment employing an open gap penalty of 10.0, an extend gap penaltyof 0.1, and using a Gonnet similarity matrix (MACVECTOR™ 10.0.2,MacVector Inc., 2008).

The term “isolated”, as used herein, refers to a substance and/or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in natureand/or in an experimental setting), and/or (2) designed, produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 400%, about 500%, about 600%, about 70%, about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or more than about 99% of the othercomponents with which they were initially associated. In someembodiments, isolated agents are about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% pure. As used herein, asubstance is “pure” if it is substantially free of other components. Insome embodiments, as will be understood by those skilled in the art, asubstance may still be considered “isolated” or even “pure”, afterhaving been combined with certain other components such as, for example,one or more carriers or excipients (e.g., buffer, solvent, water, etc.);in such embodiments, percent isolation or purity of the substance iscalculated without including such carriers or excipients. To give butone example, in some embodiments, a biological polymer such as apolypeptide or polynucleotide that occurs in nature is considered to be“isolated” when, a) by virtue of its origin or source of derivation isnot associated with some or all of the components that accompany it inits native state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; c) is expressed by or is otherwise in associationwith components from a cell or other expression system that is not ofthe species that produces it in nature. Thus, for instance, in someembodiments, a polypeptide that is chemically synthesized or issynthesized in a cellular system different from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

The phrase “non-human animal” as used herein refers to any vertebrateorganism that is not a human. In some embodiments, a non-human animal isacyclostome, a bony fish, a cartilaginous fish (e.g., a shark or a ray),an amphibian, a reptile, a mammal, and a bird. In some embodiments, anon-human mammal is a primate, a goat, a sheep, a pig, a dog, a cow, ora rodent. In some embodiments, a non-human animal is a rodent such as arat or a mouse.

The phrase “nucleic acid”, as used herein, in its broadest sense, refersto any compound and/or substance that is or can be incorporated into anoligonucleotide chain. In some embodiments, a nucleic acid is a compoundand/or substance that is or can be incorporated into an oligonucleotidechain via a phosphodiester linkage. As will be clear from context, insome embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g., nucleotides and/or nucleosides); in some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. In some embodiments, a “nucleic acid” is orcomprises RNA; in some embodiments, a “nucleic acid” is or comprisesDNA. In some embodiments, a nucleic acid is, comprises, or consists ofone or more natural nucleic acid residues. In some embodiments, anucleic acid is, comprises, or consists of one or more nucleic acidanalogs. In some embodiments, a nucleic acid analog differs from anucleic acid in that it does not utilize a phosphodiester backbone. Forexample, in some embodiments, a nucleic acid is, comprises, or consistsof one or more “peptide nucleic acids”, which are known in the art andhave peptide bonds instead of phosphodiester bonds in the backbone, areconsidered within the scope of the present invention. Alternatively oradditionally, in some embodiments, a nucleic acid has one or morephosphorothioate and/or 5′-N-phosphoramidite linkages rather thanphosphodiester bonds. In some embodiments, a nucleic acid is, comprises,or consists of one or more natural nucleosides (e.g., adenosine,thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine). In some embodiments, a nucleic acidis, comprises, or consists of one or more nucleoside analogs (e.g.,2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine,methylated bases, intercalated bases, and combinations thereof). In someembodiments, a nucleic acid comprises one or more modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) ascompared with those in natural nucleic acids. In some embodiments, anucleic acid has a nucleotide sequence that encodes a functional geneproduct such as an RNA or protein. In some embodiments, a nucleic acidincludes one or more introns. In some embodiments, nucleic acids areprepared by one or more of isolation from a natural source, enzymaticsynthesis by polymerization based on a complementary template (in vivoor in vitro), reproduction in a recombinant cell or system, and chemicalsynthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residueslong. In some embodiments, a nucleic acid is single stranded; in someembodiments, a nucleic acid is double stranded. In some embodiments anucleic acid has a nucleotide sequence comprising at least one elementthat encodes, or is the complement of a sequence that encodes, apolypeptide. In some embodiments, a nucleic acid has enzymatic activity.

The phrase “operably linked”, as used herein, refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A control sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences. “Operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest. The term “expression control sequence” asused herein refers to polynucleotide sequences which are necessary toeffect the expression and processing of coding sequences to which theyare ligated. Expression control sequences include appropriatetranscription initiation, termination, promoter and enhancer sequences;efficient RNA processing signals such as splicing and polyadenylationsignals; sequences that stabilize cytoplasmic mRNA; sequences thatenhance translation efficiency (i.e., Kozak consensus sequence);sequences that enhance protein stability; and when desired, sequencesthat enhance protein secretion. The nature of such control sequencesdiffers depending upon the host organism. For example, in prokaryotes,such control sequences generally include promoter, ribosomal bindingsite, and transcription termination sequence, while in eukaryotes,typically, such control sequences include promoters and transcriptiontermination sequence. The term “control sequences” is intended toinclude components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polypeptide”, as used herein, refers to any polymeric chain ofamino acids. In some embodiments, a polypeptide has an amino acidsequence that occurs in nature. In some embodiments, a polypeptide hasan amino acid sequence that does not occur in nature. In someembodiments, a polypeptide has an amino acid sequence that is engineeredin that it is designed and/or produced through action of the hand ofman.

The term “recombinant”, as used herein, is intended to refer topolypeptides (e.g., signal-regulatory proteins as described herein) thatare designed, engineered, prepared, expressed, created or isolated byrecombinant means, such as polypeptides expressed using a recombinantexpression vector transfected into a host cell, polypeptides isolatedfrom a recombinant, combinatorial human polypeptide library (HoogenboomH. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002)Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597, LittleM. et al (2000) Immunology Today 21:364-370) or polypeptides prepared,expressed, created or isolated by any other means that involves splicingselected sequence elements to one another. In some embodiments, one ormore of such selected sequence elements is found in nature. In someembodiments, one or more of such selected sequence elements is designedin silico. In some embodiments, one or more such selected sequenceelements results from mutagenesis (e.g., in vivo or in vitro) of a knownsequence element, e.g., from a natural or synthetic source. For example,in some embodiments, a recombinant polypeptide is comprised of sequencesfound in the genome of a source organism of interest (e.g., human,mouse, etc.). In some embodiments, a recombinant polypeptide has anamino acid sequence that resulted from mutagenesis (e.g., in vitro or invivo, for example in a non-human animal), so that the amino acidsequences of the recombinant polypeptides are sequences that, whileoriginating from and related to polypeptides sequences, may notnaturally exist within the genome of a non-human animal in vivo.

The term “replacement” is used herein to refer to a process throughwhich a “replaced” nucleic acid sequence (e.g., a gene) found in a hostlocus (e.g., in a genome) is removed from that locus and a different,“replacement” nucleic acid is located in its place. In some embodiments,the replaced nucleic acid sequence and the replacement nucleic acidsequences are comparable to one another in that, for example, they arehomologous to one another and/or contain corresponding elements (e.g.,protein-coding elements, regulatory elements, etc.). In someembodiments, a replaced nucleic acid sequence includes one or more of apromoter, an enhancer, a splice donor site, a splice receiver site, anintron, an exon, an untranslated region (UTR); in some embodiments, areplacement nucleic acid sequence includes one or more coding sequences.In some embodiments, a replacement nucleic acid sequence is a homolog ofthe replaced nucleic acid sequence. In some embodiments, a replacementnucleic acid sequence is an ortholog of the replaced sequence. In someembodiments, a replacement nucleic acid sequence is or comprises a humannucleic acid sequence. In some embodiments, including where thereplacement nucleic acid sequence is or comprises a human nucleic acidsequence, the replaced nucleic acid sequence is or comprises a rodentsequence (e.g., a mouse sequence).

The nucleic acid sequence so placed may include one or more regulatorysequences that are part of source nucleic acid sequence used to obtainthe sequence so placed (e.g., promoters, enhancers, 5′- or3′-untranslated regions, etc.). For example, in various embodiments, thereplacement is a substitution of an endogenous sequence with aheterologous sequence that results in the production of a gene productfrom the nucleic acid sequence so placed (comprising the heterologoussequence), but not expression of the endogenous sequence; thereplacement is of an endogenous genomic sequence with a nucleic acidsequence that encodes a protein that has a similar function as a proteinencoded by the endogenous sequence (e.g., the endogenous genomicsequence encodes a SIRPα protein, and the DNA fragment encodes one ormore human SIRPα proteins). In various embodiments, an endogenous geneor fragment thereof is replaced with a corresponding human gene orfragment thereof. A corresponding human gene or fragment thereof is ahuman gene or fragment that is an ortholog of, or is substantiallysimilar or the same in structure and/or function, as the endogenous geneor fragment thereof that is replaced.

The phrase “signal-regulatory protein” or “SIRP” as used herein refersto a signal-regulatory protein receptor, e.g., a SIRPα receptor. SIRPgenes include a plasma membrane receptor that is expressed on thesurface of a cell and serves as a regulatory protein involved ininteractions between membrane surface proteins on leukocytes. Within theSIRP genes, polymorphic variants have been described in human subjects.By way of illustration, nucleotide and amino acid sequences of a humanand mouse SIRP genes are provided in Table 1. Persons of skill uponreading this disclosure will recognize that one or more endogenous SIRPreceptor genes in a genome (or all) can be replaced by one or moreheterologous SIRP genes (e.g., polymorphic variants, subtypes ormutants, genes from another species, humanized forms, etc.).

A “SIRP-expressing cell” as used herein refers to a cell that expressesa signal-regulatory protein receptor. In some embodiments, aSIRP-expressing cell expresses a signal-regulatory protein receptor onits surface. In some embodiments, a SIRP protein expressed on thesurface of the cell in an amount sufficient to mediate cell-to-cellinteractions via the SIRP protein expressed on the surface of the cell.Exemplary SIRP-expressing cells include neurons, lymphocytes, myeloidcells, macrophages, neutrophils, and natural killer (NK) cells.SIRP-expressing cells regulate the interaction of immune cells toregulate the immune response to various foreign antigens or pathogens.In some embodiments, non-human animals of the present inventiondemonstrate immune cell regulation via humanized SIRP receptorsexpressed on the surface of one more cells of the non-human animal.

The term “substantially” as used herein refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena.

The phrase “substantial homology” as used herein refers to a comparisonbetween amino acid or nucleic acid sequences. As will be appreciated bythose of ordinary skill in the art, two sequences are generallyconsidered to be “substantially homologous” if they contain homologousresidues in corresponding positions. Homologous residues may beidentical residues. Alternatively, homologous residues may benon-identical residues will appropriately similar structural and/orfunctional characteristics. For example, as is well known by those ofordinary skill in the art, certain amino acids are typically classifiedas “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar”or “non-polar” side chains. Substitution of one amino acid for anotherof the same type may often be considered a “homologous” substitution.Typical amino acid categorizations are summarized in Table 1 and 2.

TABLE 1 Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive−4.5 Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polarnegative −3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu Epolar negative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly Gnonpolar neutral −0.4 Histidine His H polar positive −3.2 Isoleucine IleI nonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2

TABLE 2 Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or asparticacid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa X

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 9, 10, 11, 12, 13, 14, 15, 16, 17 or moreresidues.

In some embodiments, the relevant stretch includes contiguous residuesalong a complete sequence. In some embodiments, the relevant stretchincludes discontinuous residues along a complete sequence. In someembodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35,40, 45, 50, or more residues.

The phrase “substantial identity” as used herein refers to a comparisonbetween amino acid or nucleic acid sequences. As will be appreciated bythose of ordinary skill in the art, two sequences are generallyconsidered to be “substantially identical” if they contain identicalresidues in corresponding positions. As is well known in this art, aminoacid or nucleic acid sequences may be compared using any of a variety ofalgorithms, including those available in commercial computer programssuch as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, andPSI-BLAST for amino acid sequences. Exemplary such programs aredescribed in Altschul, et al., Basic local alignment search tool, J.Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods inEnzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997;Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis ofGenes and Proteins, Wiley, 1998; and Misener, et al., (eds.),Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol.132), Humana Press, 1999. In addition to identifying identicalsequences, the programs mentioned above typically provide an indicationof the degree of identity. In some embodiments, two sequences areconsidered to be substantially identical if at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore of their corresponding residues are identical over a relevantstretch of residues. In some embodiments, the relevant stretch is acomplete sequence. In some embodiments, the relevant stretch is at least10, 15, 20, 25, 30, 35, 40, 45, 50, or more residues.

The phrase “targeting vector” or “targeting construct” as used hereinrefers to a polynucleotide molecule that comprises a targeting region. Atargeting region comprises a sequence that is identical or substantiallyidentical to a sequence in a target cell, tissue or animal and providesfor integration of the targeting construct into a position within thegenome of the cell, tissue or animal via homologous recombination.Targeting regions that target using site-specific recombinaserecognition sites (e.g., loxP or Frt sites) are also included. In someembodiments, a targeting construct of the present invention furthercomprises a nucleic acid sequence or gene of particular interest, aselectable marker, control and or regulatory sequences, and othernucleic acid sequences that allow for recombination mediated throughexogenous addition of proteins that aid in or facilitate recombinationinvolving such sequences. In some embodiments, a targeting construct ofthe present invention further comprises a gene of interest in whole orin part, wherein the gene of interest is a heterologous gene thatencodes a protein in whole or in part that has a similar function as aprotein encoded by an endogenous sequence.

The term “variant”, as used herein, refers to an entity that showssignificant structural identity with a reference entity but differsstructurally from the reference entity in the presence or level of oneor more chemical moieties as compared with the reference entity. In manyembodiments, a variant also differs functionally from its referenceentity. In general, whether a particular entity is properly consideredto be a “variant” of a reference entity is based on its degree ofstructural identity with the reference entity. As will be appreciated bythose skilled in the art, any biological or chemical reference entityhas certain characteristic structural elements. A variant, bydefinition, is a distinct chemical entity that shares one or more suchcharacteristic structural elements. To give but a few examples, a smallmolecule may have a characteristic core structural element (e.g., amacrocycle core) and/or one or more characteristic pendent moieties sothat a variant of the small molecule is one that shares the corestructural element and the characteristic pendent moieties but differsin other pendent moieties and/or in types of bonds present (single vs.double, E vs. Z, etc) within the core, a polypeptide may have acharacteristic sequence element comprised of a plurality of amino acidshaving designated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular biologicalfunction, a nucleic acid may have a characteristic sequence elementcomprised of a plurality of nucleotide residues having designatedpositions relative to on another in linear or three-dimensional space.For example, a variant polypeptide may differ from a referencepolypeptide as a result of one or more differences in amino acidsequence and/or one or more differences in chemical moieties (e.g.,carbohydrates, lipids, etc) covalently attached to the polypeptidebackbone. In some embodiments, a variant polypeptide shows an overallsequence identity with a reference polypeptide that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.Alternatively or additionally, in some embodiments, a variantpolypeptide does not share at least one characteristic sequence elementwith a reference polypeptide. In some embodiments, the referencepolypeptide has one or more biological activities. In some embodiments,a variant polypeptide shares one or more of the biological activities ofthe reference polypeptide. In some embodiments, a variant polypeptidelacks one or more of the biological activities of the referencepolypeptide. In some embodiments, a variant polypeptide shows a reducedlevel of one or more biological activities as compared with thereference polypeptide. In many embodiments, a polypeptide of interest isconsidered to be a “variant” of a parent or reference polypeptide if thepolypeptide of interest has an amino acid sequence that is identical tothat of the parent but for a small number of sequence alterations atparticular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted ascompared with the parent. In some embodiments, a variant has 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent.Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2,or 1) number of substituted functional residues (i.e., residues thatparticipate in a particular biological activity). Furthermore, a varianttypically has not more than 5, 4, 3, 2, or 1 additions or deletions, andoften has no additions or deletions, as compared with the parent.Moreover, any additions or deletions are typically fewer than about 25,about 20, about 19, about 18, about 17, about 16, about 15, about 14,about 13, about 10, about 9, about 8, about 7, about 6, and commonly arefewer than about 5, about 4, about 3, or about 2 residues. In someembodiments, the parent or reference polypeptide is one found in nature.As will be understood by those of ordinary skill in the art, a pluralityof variants of a particular polypeptide of interest may commonly befound in nature, particularly when the polypeptide of interest is aninfectious agent polypeptide.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it is associated.In some embodiment, vectors are capable of extra-chromosomal replicationand/or expression of nucleic acids to which they are linked in a hostcell such as a eukaryotic and/or prokaryotic cell. Vectors capable ofdirecting the expression of operatively linked genes are referred toherein as “expression vectors.”

The term “wild-type”, as used herein, has its art-understood meaningthat refers to an entity having a structure and/or activity as found innature in a “normal” (as contrasted with mutant, diseased, altered, etc)state or context. Those of ordinary skill in the art will appreciatethat wild type genes and polypeptides often exist in multiple differentforms (e.g., alleles).

DETAILED DESCRIPTION

The present invention provides, among other things, improved and/orengineered non-human animals having humanized genetic material encodinga signal-regulatory protein (e.g., SIRPs) for assays in transplantengraftment, activation of phagocytosis and signal transduction. It iscontemplated that such non-human animals provides an improvement intransplant engraftment of human cells. Therefore, the present inventionis particularly useful for maintaining human hematopoietic cells innon-human animals. In particular, the present invention encompasses thehumanization of a rodent SIRPα gene resulting in expression of ahumanized protein on the plasma membrane surface of cells of thenon-human animal. Such humanized proteins have the capacity to recognizeengrafted human cells via engagement of humanized SIRPα proteins andligands present on the surface of the engrafted human cells. In someembodiments, non-human animals of the present invention are capable ofreceiving transplanted human hematopoietic cells; in some embodiments,such non-human mammals develop and/or have an immune system comprisinghuman cells. In some embodiments, humanized SIRPα proteins have sequencecorresponding to amino acid residues 28-362 of a human SIRPα protein. Insome embodiments, non-human animals of the present invention comprise anendogenous SIRPα gene that contains genetic material from the non-humananimal and a heterologous species (e.g., a human). In some embodiments,non-human animals of the present invention comprise a humanized SIRPαgene, wherein the humanized SIRPα gene comprises exons 2, 3, and 4 of ahuman SIRPα gene.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of“or” means “and/or” unless stated otherwise.

Signal-Regulatory Protein (SIRP) Gene Family

Signal regulatory proteins (SIRPs) constitute a family of cell surfaceglycoproteins which are expressed on lymphocytes, myeloid cells(including macrophages, neutrophils, granulocytes, myeloid dendriticcells, and mast cells) and neurons (e.g., see Barclay and Brown, 2006,Nat Rev Immunol 6, 457-464). There are several reported SIRP genes andthey can be categorized by their respective ligands and types ofsignaling in which they are involved. SIRPα (also referred to as CD172A,SHPS1, P84, MYD-1, BIT and PTPNS1) is expressed on immune cells of themyeloid lineage and functions as an inhibitory receptor via animmunoreceptor tyrosine-based inhibitory motif (ITIM). SIRPα expressionhas also been observed on neurons. Reported ligands for SIRPα include,most notably, CD47, but also include surfactant proteins A and D. SIRPβ(also referred to as CD172b) is expressed on macrophages andneutrophils, however, no known ligands have been reported. SIRPβcontains a short cytoplasmic region in comparison to SIRPα and is knownto associate with a signaling component known as DNAX activation protein12 (DAP12). Thus, SIRPβ is thought to be an activating receptor. SIRPγ(also referred to as CD172g and SIRPβ2) is expressed on lymphocytes andnatural killer cells and also binds to CD47, however, no signalingfunction has been reported as the cytoplasmic tail only contains fouramino acids and lacks a sequence that would facilitate association withDAP12. Another member, SIRP8, has been described and exists as a solublereceptor.

The role of SIRPα, in particular, has been investigated in respect ofits inhibitory role in the phagocytosis of host cells by macrophages.For example, CD47 binding to SIRPα on macrophages, triggers inhibitorysignals that negatively regulates phagocytosis. Alternatively, positivesignaling effects mediated through SIRPα binding have been reported(Shultz et al., 1995, J Immunol 154, 180-91).

SIRPα Sequences

Exemplary SIRPα sequences for human and mouse are set forth in Table 3.For cDNA sequences, consecutive exons are separated by alternatingunderlined text. For protein sequences, signal peptides are underlinedand transmembrane and cytoplasmic sequences are italicized.

TABLE 3 mouse SIRPα GCGCTCGGCCGGGCCGCCCTCGCGCTGGCCTCGCGACGGCTC cDNACGCACAGCCCGCACTCGCTCTGCGAGCTGTCCCCGCTCGCGCT NM_907547.3TGCTCTCCGATCTCCGTCCCCGCTCCCTCTCCCTCTTCCTCTCCCCCTCTTTCCTTCTCCCTCGCTATCCGCTCCCCCGCCCCCGTGCCTCTGGCTCTGCGCCTGGCTCCCTCGGGTCCGCTCCCCTTTCCCGCCGGCCTGGCCCGGCGTCACGCTCCCGGAGTCTCCCCGCTCGGCGGCGTCTCATTGTGGGAGGGGGTCAGATCACCCCGCCGGGCGGTGGCGCTGGGGGGCAGCGGAGGGGGAGGGGCCTTAGTCGTTCGCCCGCGCCGCCCGCCCGCCTGCCGAGCGCGCTCACCGCCGCTCTCCCTCCTTGCTCTGCAGCCGCGGCCCATGGAGCCCGCCGGCCCGGCCCCTGGCCGCCTAGGGCCGCTGCTGCTCTGCCTGCTGCTCTCCGCGTCCTGTTTCTGTACAGGAGCCACGGGGAAGGAACTGAAGGTGACTCAGCCTGAGAAATCAGTGTCTGTTGCTGCTGGGGATTCGACCGTTCTGAACTGCACTTTGACCTCCTTGTTGCCGGTGGGACCCATTAGGTGGTACAGAGGAGTAGGGCCAAGCCGGCTGTTGATCTACAGTTTCGCAGGAGAATACGTTCCTCGAATTAGAAATGTTTCAGATACTACTAAGAGAAACAATATGGACTTTTCCATCCGTATCAGTAATGTCACCCCAGCAGATGCTGGCATCTACTACTGTGTGAAGTTCCAGAAAGGATCATCAGAGCCTGACACAGAAATACAATCTGGAGGGGGAACAGAGGTCTATGTACTCGCCAAACCTTCTCCACCGGAGGTATCCGGCCCAGCAGACAGGGGCATACCTGACCAGAAAGTGAACTTCACCTGCAAGTCTCATGGCTTCTCTCCCCGGAATATCACCCTGAAGTGGTTCAAAGATGGGCAAGAACTCCACCCCTTGGAGACCACCGTGAACCCTAGTGGAAAGAATGTCTCCTACAACATCTCCAGCACAGTCAGGGTGGTACTAAACTCCATGGATGTTAATTCTAAGGTCATCTGCGAGGTAGCCCACATCACCTTGGATAGAAGCCCTCTTCGTGGGATTGCTAACCTGTCTAACTTCATCCGAGTTTCACCCACCGTGAAGGTCACCCAACAGTCCCCGACGTCAATGAACCAGGTGAACCTCACCTGCCGGGCTGAGAGGTTCTACCCCGAGGATCTCCAGCTGATCTGGCTGGAGAATGGAAACGTATCACGGAATGACACGCCCAAGAATCTCACAAAGAACACGGATGGGACCTATAATTACACAAGCTTGTTCCTGGTGAACTCATCTGCTCATAGAGAGGACGTGGTGTTCACGTGCCAGGTGAAGCACGACCAACAGCCAGCGATCACCCGAAACCATACCGTGCTGGGATTTGCCCACTCGAGTGATCAAGGGAGCATGCAAACCTTCCCTGATAATAATGCTACCCACAACTGGAATGTCTTCATCGGTGTGGGCGTGGCGTGTGCTTTGCTCGTAGTCCTGCTGATGGCTGCTCTCTACCTCCTCCGGATCAAACAGAAGAAAGCCAAGGGGTCAACATCTTCCACACGGTTGCACGAGCCCGAGAAGAACGCCAGGGAAATAACCCAGATCCAGGACACAAATGACATCAACGACATCACATACGCAGACCTGAATCTGCCCAAAGAGAAGAAGCCCGCACCCCGGGCCCCTGAGCCTAACAACCACACAGAATATGCAAGCATTGAGACAGGCAAAGTGCCTAGGCCAGAGGATACCCTCACCTATGCTGACCTGGACATGGTCCACCTCAGCCGGGCACAGCCAGCCCCCAAGCCTGAGCCATCTTTCTCAGAGTATGCTAGTGTCCAGGTCCAGAGGAAGTGAATGGGGCTGTGGTCTGTACTAGGCCCCATCCCCACAAGTTTTCTTGTCCTACATGGAGTGGCCATGACGAGGACATCCAGCCAGCCAATCCTGTCCCCAGAAGGCCAGGTGGCACGGGTCCTAGGACCAGGGGTAAGGGTGGCCTTTGTCTTCCCTCCGTGGCTCTTCAACACCTCTTGGGCACCCACGTCCCCTTCTTCCGGAGGCTGGGTGTTGCAGAACCAGAGGGCGAACTGGAGAAAGCTGCCTGGAATCCAAGAAGTGTTGTGCCTCGGCCCATCACTCGTGGGTCTGGATCCTGGTCTTGGCAACCCCAGGTTGCGTCCTTGATGTTCCAGAGCTTGGTCTTCTGTGTGGAGAAGAGCTCACCATCTCTACCCAACTTGAGCTTTGGGACCAGACTCCCTTTAGATCAAACCGCCCCATCTGTGGAAGAACTACACCAGAAGTCAGCAAGTTTTCAGCCAACAGTGCTGGCCTCCCCACCTCCCAGGCTGACTAGCCCTGGGGAGAAGGAACCCTCTCCTCCTAGACCAGCAGAGACTCCCTGGGCATGTTCAGTGTGGCCCCACCTCCCTTCCAGTCCCAGCTTGCTTCCTCCAGCTAGCACTAACTCAGCAGCATCGCTCTGTGGACGCCTGTAAATTATTGAGAAATGTGAACTGTGCAGTCTTAAAGCTAAGGTGTTAGAAAATTTGATTTATGCTGTTTAGTTGTTGTTGGGTTTCTTTTCTTTTTAATTTCTTTTTCTTTTTTGATTTTTTTTCTTTCCCTTAAAACAACAGCAGCAGCATCTTGGCTCTTTGTCATGTGTTGAATGGTTGGGTCTTGTGAAGTCTGAGGTCTAACAGTTTATTGTCCTGGAAGGATTTTCTTACAGCAGAAACAGATTTTTTTCAAATTCCCAGAATCCTGAGGACCAAGAAGGATCCCTCAGCTGCTACTTCCAGCACCCAGCGTCACTGGGACGAACCAGGCCCTGTTCTTACAAGGCCACATGGCTGGCCCTTTGCCTCCATGGCTACTGTGGTAAGTGCAGCCTTGTCTGACCCAATGCTGACCTAATGTTGGCCATTCCACATTGAGGGGACAAGGTCAGTGATGCCCCCCCTTCACTCACAAGCACTTCAGAGGCATGCAGAGAGAAGGGACACTCGGCCAGCTCTCTGAGGTAATCAGTGCAAGGAGGAGTCCGTTTTTTGCCAGCAAACCTCAGCAGGATCACACTGGAACAGAACCTGGTCATACCTGTGACAACACAGCTGTGAGCCAGGGCAAACCACCCACTGTCACTGGCTCGAGAGTCTGGGCAGAGGCTCTGACCCTCCACCCTTTAAACTGGATGCCGGGGCCTGGCTGGGCCCAATGCCAAGTGGTTATGGCAACCCTGACTATCTGGTCTTAACATGTAGCTCAGGAAGTGGAGGCGCTAATGTCCCCAATCCCTGGGGATTCCTGATTCCAGCTATTCATGTAAGCAGAGCCAACCTGCCTATTTCTGTAGGTGCGACTGGGATGTTAGGAGCACAGCAAGGACCCAGCTCTGTAGGGCTGGTGACCTGATACTTCTCATAATGGCATCTAGAAGTTAGGCTGAGTTGGCCTCACTGGCCCAGCAAACCAGAACTTGTCTTTGTCCGGGCCATGTTCTTGGGCTGTCTTCTAATTCCAAAGGGTTGGTTGGTAAAGCTCCACCCCCTTCTCCTCTGCCTAAAGACATCACATGTGTATACACACACGGGTGTATAGATGAGTTAAAAGAATGTCCTCGCTGGCATCCTAATTTTGTCTTAAGTTTTTTTTGGAGGGAGAAAGGAACAAGGCAAGGGAAGATGTGTAGCTTTGGCTTTAACCAGGCAGCCTGGGGGCTCCCAAGCCTATGGAACCCTGGTACAAAGAAGAGAACAGAAGCGCCCTGTGAGGAGTGGGATTTGTTTTTCTGTAGACCAGATGAGAAGGAAACAGGCCCTGTTTTGTACATAGTTGCAACTTAAAATTTTTGGCTTGCAAAATATTTTTGTAATAAAGATTTCTGGGTAACAATAAAAAAAAAAA AAAAAA (SEQ ID NO: 1)Mouse SIRPα MEPAGPAPGRLGPLLLCLLLSASCFCTGATGKELKVTQPEKSVSV ProteinAAGDSTVLNCTLTSLLPVGPIRWYRGVGPSRLLIYSFAGEYVPRI NP_031573.2RNVSDTTKRNNMDFSIRISNVTPADAGIYYCVKFQKGSSEPDTEIQSGGGTEVYVLAKPSPPEVSGPADRGIPDQKVNFTCKSHGFSPRNITLKWFKDGQELHPLETTVNPSGKNVSYNISSTVRVVLNSMDVNSKVICEVAHITLDRSPLRGIANLSNFIRVSPTVKVTQQSPTSMNQVNLTCRAERTYPEDLQLIWLENGNVSRNDTPKNLTKNTDGTYNYTSLFLVNSSAHREDVVFTCQVKHDQQPAITRNHTVLGFAHSSDQGSMQTFPDNNATHNWNVFIGVGVACALLVVLLMAALYLLRIKQKKAKGSTSSTRLHEPEKNAREITQIQDTNDINDITYADLNLPKEKKPAPRAPEPNNHTEYASIETGKVPRPEDTLTYADLDMVHLSRAQPAPKPEPSFS EYSVQVQRK (SEQ IN NO: 2)Human SIRPα TCCGGCCCGCACCCACCCCCAAGAGGGGCCTTCAGCTTTGGG DNA GCTCAGAGGCACGACCTCCTGGGGAGGGTTAAAAGGCAGACG NM_001040022.1CCCCCCCGCCCCCCGCGCCCCCGCGCCCCCGACTCCTTCGCCGCCTCCAGCCTCTCGCCAGTGGGAAGCGGGGAGCAGCCGCGCGGCCGGAGTCCGGAGGCGAGGGGAGGTCGGCCGCAACTTCCCCGGTCCACCTTAAGAGGACGATGTAGCCAGCTCGCAGCGCTGACCTTAGAAAAACAAGTTTGCGCAAAGTGGAGCGGGGACCCGGCCTCTGGGCAGCCCCCGGCGGCGCTTCCAGTGCCTTCCAGCCCTCGCGGGCGGCGCAGCCGCGGCCCATGGAGCCCGCCGGCCCGGCCCCCGGCCGCCTCGGGCCGCTGCTCTGCCTGCTGCTCGCCGCGTCCTGCGCCTGGTCAGGAGTGGCGGGTGAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAGCCACTCTGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGGACCAGGCCGGGAATTAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTTTCAGACCTCACAAAGAGAAACAACATGGACTTTTCCATCCGCATCGGTAACATCACCCCAGCAGATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGAGCCCCGATGACGTGGAGTTTAAGTCTGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGGTATCGGGCCCTGCGGCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCAGAGACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGTAGGAGAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGACGTTCACTCTCAAGTCATCTGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTGCCAACTTGTCTGAGACCATCCGAGTTCCACCCACCTTGGAGGTTACTCAACAGCCCGTGAGGGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAGTTCTACCCCCAGAGACTACAGCTGACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTTACAGAGAACAAGGATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTGAAGCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCAGCCCACCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGGTGGCCCTACTGATGGCGGCCCTCTACCTCGTCCGAATCAGACAGAAGAAAGCCCAGGGCTCCACTTCTTCTACAAGGTTGCATGAGCCCGAGAAGAATGCCAGAGAAATAACACAGGACACAAATGATATCACATATGCAGACCTGAACCTGCCCAAGGGGAAGAAGCCTGCTCCCCAGGCTGCGGAGCCCAACAACCACACGGAGTATGCCAGCATTCAGACCAGCCCGCAGCCCGCGTCGGAGGACACCCTCACCTATGCTGACCTGGACATGGTCCACCTCAACCGGACCCCCAAGCAGCCGGCCCCCAAGCCTGAGCCGTCCTTCTCAGAGTACGCCAGCGTCCAGGTCCCGAGGAAGTGAATGGGACCGTGGTTTGCTCTAGCACCCATCTCTACGCGCTTTCTTGTCCCACAGGGAGCCGCCGTGATGAGCACAGCCAACCCAGTTCCCGGAGGGCTGGGGCGGTGCAGGCTCTGGGACCCAGGGGCCAGGGTGGCTCTTCTCTCCCCACCCCTCCTTGGCTCTCCAGCACTTCCTGGGCAGCCACGGCCCCCTCCCCCCACATTGCCACATACCTGGAGGCTGACGTTGCCAAACCAGCCAGGGAACCAACCTGGGAAGTGGCCAGAACTGCCTGGGGTCCAAGAACTCTTGTGCCTCCGTCCATCACCATGTGGGTTTTGAAGACCCTCGACTGCCTCCCCGATGCTCCGAAGCCTGATCTTCCAGGGTGGGGAGGAGAAAATCCCACCTCCCCTGACCTCCACCACCTCCACCACCACCACCACCACCACCACCACCACTACCACCACCACCCAACTGGGGCTAGAGTGGGGAAGATTTCCCCTTTAGATCAAACTGCCCCTTCCATGGAAAAGCTGGAAAAAAACTCTGGAACCCATATCCAGGCTTGGTGAGGTTGCTGCCAACAGTCCTGGCCTCCCCCATCCCTAGGCTAAAGAGCCATGAGTCCTGGAGGAGGAGAGGACCCCTCCCAAAGGACTGGAGACAAAACCCTCTGCTTCCTTGGGTCCCTCCAAGACTCCCTGGGGCCCAACTGTGTTGCTCCACCCGGACCCATCTCTCCCTTCTAGACCTGAGCTTGCCCCTCCAGCTAGCACTAAGCAACATCTCGCTGTGGACGCCTGTAAATTACTGAGAAATGTGAAACGTGCAATCTTGAAACTGAGGTGTTAGAAAACTTGATCTGTGGTGTTTTGTTTTGTTTTTTTTCTTAAAACAACAGCAACGTGATCTTGGCTGTCTGTCATGTGTTGAAGTCCATGGTTGGGTCTTGTGAAGTCTGAGGTTTAACAGTTTGTTGTCCTGGAGGGATTTTCTTACAGCGAAGACTTGAGTTCCTCCAAGTCCCAGAACCCCAAGAATGGGCAAGAAGGATCAGGTCAGCCACTCCCTGGAGACACAGCCTTCTGGCTGGGACTGACTTGGCCATGTTCTCAGCTGAGCCACGCGGCTGGTAGTGCAGCCTTCTGTGACCCCGCTGTGGTAAGTCCAGCCTGCCCAGGGCTGCTGAGGGCTGCCTCTTGACAGTGCAGTCTTATCGAGACCCAATGCCTCAGTCTGCTCATCCGTAAAGTGGGGATAGTGAAGATGACACCCCTCCCCACCACCTCTCATAAGCACTTTAGGAACACACAGAGGGTAGGGATAGTGGCCCTGGCCGTCTATCCTACCCCTTTAGTGACCGCCCCCATCCCGGCTTTCTGAGCTGATCCTTGAAGAAGAAATCTTCCATTTCTGCTCTCAAACCCTACTGGGATCAAACTGGAATAAATTGAAGACAGCCAGGGGGATGGTGCAGCTGTGAAGCTCGGGCTGATTCCCCCTCTGTCCCAGAAGGTTGGCCAGAGGGTGTGACCCAGTTACCCTTTAACCCCCACCCTTCCAGTCGGGTGTGAGGGCCTGACCGGGCCCAGGGCAAGCAGATGTCGCAAGCCCTATTTATTCAGTCTTCACTATAACTCTTAGAGTTGAGACGCTAATGTTCATGACTCCTGGCCTTGGGATGCCCAAGGGATTTCTGGCTCAGGCTGTAAAAGTAGCTGAGCCATCCTGCCCATTCCTGGAGGTCCTACAGGTGAAACTGCAGGAGCTCAGCATAGACCCAGCTCTCTGGGGGATGGTCACCTGGTGATTTCAATGATGGCATCCAGGAATTAGCTGAGCCAACAGACCATGTGGACAGCTTTGGCCAGAGCTCCCGTGTGGCATCTGGGAGCCACAGTGACCCAGCCACCTGGCTCAGGCTAGTTCCAAATTCCAAAAGATTGGCTTGTAAACCTTCGTCTCCCTCTCTTTTACCCAGAGACAGCACATACGTGTGCACACGCATGCACACACACATTCAGTATTTTAAAAGAATGTTTTCTTGGTGCCATTTTCATTTTATTTTATTTTTTAATTCTTGGAGGGGGAAATAAGGGAATAAGGCCAAGGAAGATGTATAGCTTTAGCTTTAGCCTGGCAACCTGGAGAATCCACATACCTTGTGTATTGAACCCCAGGAAAAGGAAGAGGTCGAACCAACCCTGCGGAAGGAGCATGGTTTCAGGAGTTTATTTTAAGACTGCTGGGAAGGAAACAGGCCCCATTTTGTATATAGTTGCAACTTAAACTTTTTGGCTTGCAAAATATTTTTGTAATAAAGATTTCTGGGTAATAATGA (SEQ ID NO: 3) Human SIRPαMEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVL ProteinVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPR NP_001035111.1VTTVSDLTKNNMDFSIRIGNITPADAGTYYCVKIRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNNTDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKA QGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYASLSMVHLNRTPKQPAPKPEPSFSEY ASVQVPRK (SEQ ID NO: 4)Humanized MEPAGPAPGRLGPLLLCLLLSASCFCTGVAGEEELQVIQPDKSVL SMPα ProteinVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGELFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAADNNATHNWNVFIGVGVACALLVVLLMAALYLLRIKQKKAKGSTSSTRLHEPEKNAREITQIQDTADINDITYADLNLPKEKKPAPRAPEPNNHTEYASIETGKVPRPEDTLTYADLDMVHLSRAQPAPKPEPSFSEYASVQVQRK (SEQ ID NO: 5)Humanized SIRPα Non-Human Animals

Non-human animals are provided that express humanized SIRPα proteins onthe surface of immune cells (e.g., myeloid cells) of the non-humananimals resulting from a genetic modification of an endogenous locus ofthe non-human animal that encodes a SIRPα protein. Suitable examplesdescribed herein include rodents, in particular, mice.

A humanized SIRPα gene, in some embodiments, comprises genetic materialfrom a heterologous species (e.g., humans), wherein the humanized SIRPαgene encodes a SIRPα protein that comprises the encoded portion of thegenetic material from the heterologous species. In some embodiments, ahumanized SIRPα gene of the present invention comprises genomic DNA of aheterologous species that corresponds to the extracellular portion of aSIRPα protein that is expressed on the plasma membrane of a cell.Non-human animals, embryos, cells and targeting constructs for makingnon-human animals, non-human embryos, and cells containing saidhumanized SIRPα gene are also provided.

In some embodiments, an endogenous SIRPα gene is deleted. In someembodiments, an endogenous SIRPα gene is altered, wherein a portion ofthe endogenous SIRPα gene is replaced with a heterologous sequence(e.g., a human SIRPα sequence in whole or in part). In some embodiments,all or substantially all of an endogenous SIRPα gene is replaced with aheterologous gene (e.g., a human SIRPα gene). In some embodiments, aportion of a heterologous SIRPα gene is inserted into an endogenousnon-human SIRPα gene at an endogenous SIRPα locus. In some embodiments,the heterologous gene is a human gene. In some embodiments, themodification or humanization is made to one of the two copies of theendogenous SIRPα gene, giving rise to a non-human animal is heterozygouswith respect to the humanized SIRPα gene. In other embodiments, anon-human animal is provided that is homozygous for a humanized SIRPαgene.

A non-human animal of the present invention contains a human SIRPα genein whole or in part at an endogenous non-human SIRPα locus. Thus, suchnon-human animals can be described as having a heterologous SIRP gene.The replaced, inserted or modified SIRPα gene at the endogenous SIRPαlocus can be detected using a variety of methods including, for example,PCR, Western blot, Southern blot, restriction fragment lengthpolymorphism (RFLP), or a gain or loss of allele assay. In someembodiments, the non-human animal is heterozygous with respect to thehumanized SIRPα gene

In various embodiments, a humanized SIRPα gene according to the presentinvention includes a SIRPα gene that has a second, third and fourth exoneach having a sequence at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to a second, third and fourth exon that appear in a humanSIRPα gene of Table 3.

In various embodiments, a humanized SIRPα gene according to the presentinvention includes a SIRPα gene that has a nucleotide coding sequence(e.g., a cDNA sequence) at least 50% (e.g., 50%, 55%, 60%, 65%, 70%,75%, 800/0, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore) identical to nucleotides 352-1114 that appear in a human SIRPαcDNA sequence of Table 3.

In various embodiments, a humanized SIRPα protein produced by anon-human animal of the present invention has an extracellular portionhaving a sequence that is at least 500 (e.g., 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to an extracellular portion of a human SIRPα protein thatappears in Table 3.

In various embodiments, a humanized SIRPα protein produced by anon-human animal of the present invention has an extracellular portionhaving a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 700%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)identical to amino acid residues 28-362 that appear in a human SIRPαprotein of Table 3.

In various embodiments, a humanized SIRPα protein produced by anon-human animal of the present invention has an amino acid sequencethat is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 990% or more) identical to anamino acid sequence of a humanized SIRPα protein that appears in Table3.

Compositions and methods for making non-human animals that expresses ahumanized SIRPα protein, including specific polymorphic forms or allelicvariants (e.g., single amino acid differences), are provided, includingcompositions and methods for making non-human animals that expressessuch proteins from a human promoter and a human regulatory sequence. Insome embodiments, compositions and methods for making non-human animalsthat expresses such proteins from an endogenous promoter and anendogenous regulatory sequence are also provided. The methods includeinserting the genetic material encoding a human SIRPα protein in wholeor in part at a precise location in the genome of a non-human animalthat corresponds to an endogenous SIRPα gene thereby creating ahumanized SIRPα gene that expresses a SIRPα protein that is human inwhole or in part. In some embodiments, the methods include insertinggenomic DNA corresponding to exons 2-4 of a human SIRPα gene into anendogenous SIRPα gene of the non-human animal thereby creating ahumanized gene that encodes a SIRPα protein that contains a humanportion containing amino acids encoded by the inserted exons.

A humanized SIRPα gene approach employs a relatively minimalmodification of the endogenous gene and results in naturalSIRPα-mediated signal transduction in the non-human animal, in variousembodiments, because the genomic sequence of the SIRPα sequences aremodified in a single fragment and therefore retain normal functionalityby including necessary regulatory sequences. Thus, in such embodiments,the SIRPα gene modification does not affect other surrounding genes orother endogenous SIRP genes. Further, in various embodiments, themodification does not affect the assembly of a functional receptor onthe plasma and maintains normal effector functions via binding andsubsequent signal transduction through the cytoplasmic portion of thereceptor which is unaffected by the modification.

A schematic illustration (not to scale) of an endogenous murine SIRPαgene and a humanized SIRPα gene is provided in FIG. 1. As illustrated,genomic DNA containing exons 2-4 of a human SIRPα gene is inserted intoan endogenous murine SIRPα gene locus by a targeting construct. Thisgenomic DNA includes comprises the portion of the gene that encodes anextracellular portion (e.g., amino acid resides 28-362) of a human SIRPαprotein responsible for ligand binding.

A non-human animal (e.g., a mouse) having a humanized SIRPα gene at theendogenous SIRPα locus can be made by any method known in the art. Forexample, a targeting vector can be made that introduces a human SIRPαgene in whole or in part with a selectable marker gene. FIG. 1illustrates a mouse genome comprising an insertion of exons 2-4 of ahuman SIRPα. As illustrated, the targeting construct contains a 5′homology arm containing sequence upstream of exon 2 of an endogenousmurine SIRPα gene, followed by a genomic DNA fragment containing exons2-4 of a human SIRPα gene, a drug selection cassette (e.g., a neomycinresistance gene flanked on both sides by loxP sequences), and a 3′homology arm containing sequence downstream of exons 4 of an endogenousmurine SIRPα gene. Upon homologous recombination, exons 2-4 of anendogenous murine SIRPα gene is replaced by the sequence contained inthe targeting vector. A humanized SIRPα gene is created resulting in acell or non-human animal that expresses a humanized SIRPα protein thatcontains amino acids encoded by exons 2-4 of a human SIRPα gene. Thedrug selection cassette may optionally be removed by the subsequentaddition of a recombinase (e.g., by Cre treatment).

In addition to mice having humanized SIRPα genes as described herein,also provided herein are other genetically modified non-human animalsthat comprise humanized SIRPα genes. In some embodiments, such non-humananimals comprise a humanized SIRPα gene operably linked to an endogenousSIRPα promoter. In some embodiments, such non-human animals express ahumanized SIRPα protein from an endogenous locus, wherein the humanizedSIRPα protein comprises amino acid residues 28-362 of a human SIRPαprotein.

Such non-human animals may be selected from the group consisting of amouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep,goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesusmonkey). For the non-human animals where suitable genetically modifiableES cells are not readily available, other methods are employed to make anon-human animal comprising genetic modifications as described herein.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

In some embodiments, a non-human animal of the present invention is amammal. In some embodiments, a non-human animal of the present inventionis a small mammal, e.g., of the superfamily Dipodoidea or Muroidea. Insome embodiments, a genetically modified animal of the present inventionis a rodent. In some embodiments, a rodent of the present invention isselected from a mouse, a rat, and a hamster. In some embodiments, arodent of the present invention is selected from the superfamilyMuroidea. In some embodiments, a genetically modified animal of thepresent invention is from a family selected from Calomyscidae (e.g.,mouse-like hamsters), Cricetidae (e.g., hamster, New World rats andmice, voles), Muridae (true mice and rats, gerbils, spiny mice, crestedrats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some certainembodiments, a genetically modified rodent of the present invention isselected from a true mouse or rat (family Muridae), a gerbil, a spinymouse, and a crested rat. In some certain embodiments, a geneticallymodified mouse of the present invention is from a member of the familyMuridae. In some embodiment, an non-human animal of the presentinvention is a rodent. In some certain embodiments, a rodent of thepresent invention is selected from a mouse and a rat. In someembodiments, a non-human animal of the present invention is a mouse.

In some embodiments, a non-human animal of the present invention is arodent that is a mouse of a C57BL strain selected from C57BL/A,C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BUL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In somecertain embodiments, a mouse of the present invention is a 129 strainselected from the group consisting of a strain that is 129P1, 129P2,129P3,129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5,129S9/SvEvH, 129/SvJae, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2(see, e.g., Festing et al., 1999, Mammalian Genome 10:836; Auerbach etal., 2000, Biotechniques 29(5):1024-1028, 1030, 1032). In some certainembodiments, a genetically modified mouse of the present invention is amix of an aforementioned 129 strain and an aforementioned C57BL/6strain. In some certain embodiments, a mouse of the present invention isa mix of aforementioned 129 strains, or a mix of aforementioned BL/6strains. In some certain embodiments, a 129 strain of the mix asdescribed herein is a 129S6 (129/SvEvTac) strain. In some embodiments, amouse of the present invention is a BALB strain, e.g., BALB/c strain. Insome embodiments, a mouse of the present invention is a mix of a BALBstrain and another aforementioned strain.

In some embodiments, a non-human animal of the present invention is arat. In some certain embodiments, a rat of the present invention isselected from a Wistar rat, an LEA strain, a Sprague Dawley strain, aFischer strain, F344, F6, and Dark Agouti. In some certain embodiments,a rat strain as described herein is a mix of two or more strainsselected from the group consisting of Wistar, LEA, Sprague Dawley,Fischer, F344, F6, and Dark Agouti.

Methods Employing Non-Human Animals Having Humanized SIRPα Genes

SIRPα mutant and transgenic non-human animals (e.g., mice) have beenreported (Inagaki et al., 2000, EMBO Journal 19(24):6721-6731; Strowiget al., 2011, Proc. Nat. Acad. Sci. 108(32): 13218-13223). Such animalshave been employed in a variety of assays to determine the molecularaspects of SIRPα expression, function and regulation. However, they arenot without limitation. For example, use of SIRPα mutant mice have beenlimited due to deleterious health conditions resulting from an inabilityof cells containing the mutant form of SIRPα to signal. Further, becauseCD47, a ligand for SIRPα, might be present on the same cell as themutant form of SIRPα and both proteins are capable of providingintracellular signals, it is not possible to distinguish if such resultsare from lack of SIRPα signaling or lack of CD47 binding. In the case ofhuman SIRPα transgenic mice, mouse SIRPα is intact and functional. Thus,SIRPα-dependent functions in various biological processes (e.g.,engraftment) cannot be clearly attributed to either human SIRPα or mouseSIRPα function alone in these mice as both the human and mouse SIRPαreceptors are present and functional.

Non-human animals of the present invention provide an improved in vivosystem and source of biological materials (e.g., cells) expressing humanSIRPα that are useful for a variety of assays. In various embodiments,non-human animals of the present invention are used to developtherapeutics that target SIRPα and/or modulate SIRPα-CD47 signaling. Invarious embodiments, mice of the present invention are used to screenand develop candidate therapeutics (e.g., antibodies) that bind to humanSIRPα. In various embodiments, non-human animals of the presentinvention are used to determine the binding profile of antagonistsand/or agonists a humanized SIRPα on the surface of a cell of anon-human animal as described herein.

In various embodiments, non-human animals of the present invention areused to measure the therapeutic effect of blocking or modulating SIRPαsignal transduction (e.g., phosphorylation) and the effect on geneexpression as a result of cellular changes. In various embodiments, anon-human animal of the present invention of cells isolated therefromare exposed to a candidate therapeutic that binds to a human SIRPα onthe surface of a cell of the non-human animal and, after a subsequentperiod of time, analyzed for effects on SIRPα-dependent processes, forexample, B and/or T cell proliferation, clearance of platelets, andinduction of cytokine expression.

Non-human animals of the present invention express humanized SIRPαprotein, thus cells, cell lines, and cell cultures can be generated toserve as a source of humanized SIRPα for use in binding and functionalassays, e.g., to assay for binding or function of a SIRPα antagonist oragonist, particularly where the antagonist or agonist is specific for ahuman SIRPα sequence or epitope. In various embodiments, a humanizedSIRPα protein expressed by a non-human animal as described herein maycomprise a variant amino acid sequence. Variant human SIRPα proteinshaving variations associated with ligand binding residues have beenreported. In various embodiments, non-human animals of the presentinvention express a humanized SIRPα protein variant. In variousembodiments, the variant is polymorphic at an amino acid positionassociated with ligand binding. In various embodiments, non-humananimals of the present invention are used to determine the effect ofligand binding through interaction with a polymorphic variant of humanSIRPα.

Cells from non-human animals of the present invention can be isolatedand used on an ad hoc basis, or can be maintained in culture for manygenerations. In various embodiments, cells from a non-human animal ofthe present invention are immortalized and maintained in cultureindefinitely (e.g., in serial cultures).

In various embodiments, cells of non-human animals of the presentinvention are used in a cell migration or spreading assay to screen anddevelop candidate therapeutics that modulate human SIRPα. Such processesare necessary for many cellular processes including wound healing,differentiation, proliferation and survival.

In various embodiments, cells of non-human animals of the presentinvention are used in clonal assays for megakaryocytic colony-formingcells for testing the pharmaco-toxicological aspects of candidatetherapeutics that target human SIRPα.

In various embodiments, cells of non-human animals of the presentinvention are used in phagocytosis assays to determine the therapeuticpotential of compounds or biological agents to modulate SIRPα-dependentregulation of phagocytosis.

Non-human animals of the present invention provide an in vivo system forthe analysis and testing of a drug or vaccine. In various embodiments, acandidate drug or vaccine may be delivered to one or more non-humananimals of the present invention, followed by monitoring of thenon-human animals to determine one or more of the immune response to thedrug or vaccine, the safety profile of the drug or vaccine, or theeffect on a disease or condition. Such drugs or vaccines may be improvedand/or developed in such non-human animals.

Non-human animals of the present invention provide improved in vivosystem elucidating mechanisms of human cell-to-cell interaction throughadoptive transfer. In various embodiments, non-human animals of thepresent invention may by implanted with a tumor xenograft, followed by asecond implantation of tumor infiltrating lymphocytes could be implantedin the non-human animals by adoptive transfer to determine theeffectiveness in eradication of solid tumors or other malignancies. Suchexperiments may be done with human cells due to the exclusive presenceof human SIRPα without competition with endogenous SIRPα of thenon-human animal. Further, therapies and pharmaceuticals for use inxenotransplantation can be improved and/or developed in such non-humananimals.

Non-human animals of the present invention provide an improved in vivosystem for maintenance and development of human hematopoietic stem cellsthrough engraftment. In various embodiments, non-human animals of thepresent invention provide improved development and maintenance of humanstem cells within the non-human animal. In various embodiments,increased populations of differentiated human B and T cells are observedin the blood, bone marrow, spleen and thymus of the non-human animal. Invarious embodiments, non-human animals of the present invention providean increase in the level of engraftment of human cells as compared tonon-human animals that express both mouse and human SIRPα.

Non-human animals of the present invention can be employed to assess theefficacy of a therapeutic drug targeting human cells. In variousembodiments, a non-human animal of the present invention is transplantedwith human cells, and a drug candidate targeting such human cells isadministered to such animal. The therapeutic efficacy of the drug isthen determined by monitoring the human cells in the non-human animalafter the administration of the drug. Drugs that can be tested in thenon-human animals include both small molecule compounds, i.e., compoundsof molecular weights of less than 1500 kD, 1200 kD, 1000 kD, or 800dalton, and large molecular compounds (such as proteins, e.g.,antibodies), which have intended therapeutic effects for the treatmentof human diseases and conditions by targeting (e.g., binding to and/oracting on) human cells.

In some embodiments, the drug is an anti-cancer drug, and the humancells are cancer cells, which can be cells of a primary cancer or cellsof cell lines established from a primary cancer. In these embodiments, anon-human animal of the present invention is transplanted with humancancer cells, and an anti-cancer drug is given to the non-human animal.The efficacy of the drug can be determined by assessing whether growthor metastasis of the human cancer cells in the non-human animal isinhibited as a result of the administration of the drug.

In specific embodiments, the anti-cancer drug is an antibody moleculewhich binds to an antigen on human cancer cells. In particularembodiments, the anti-cancer drug is a bispecific antibody that binds toan antigen on human cancer cells, and to an antigen on other humancells, for example, cells of the human immune system (or “human immunecells”) such as B cells and T cells.

In some embodiments, a non-human animal of the present invention isengrafted with human immune cells or cells that differentiate into humanimmune cells. Such non-human animal with engrafted human immune cells istransplanted with human cancer cells, and is administered with ananti-cancer drug, such as a bispecific antibody that binds to an antigenon human cancer cells and to an antigen on human immune cells (e.g.,T-cells). The therapeutic efficacy of the bispecific antibody can beevaluated based on its ability to inhibit growth or metastasis of thehuman cancer cells in the non-human animal. In a specific embodiment,the non-human animal of the present invention is engrafted with humanCD34+ hematopoietic progenitor cells which give rise to human immunecells (including T cells, B cells, NK cells, among others). Human B celllymphoma cells (e.g., Raji cells) are transplanted into such non-humananimal with engrafted human immune cells, which is then administeredwith a bispecific antibody that binds to CD20 (an antigen on normal Bcells and certain B cell malignancies) and to the CD3 subunit of theT-cell receptor, to test the ability of the bispecific antibody toinhibit tumor growth in the non-human animal.

EXAMPLES

The following examples are provided so as to describe to those ofordinary skill in the art how to make and use methods and compositionsof the invention, and are not intended to limit the scope of what theinventors regard as their invention. Unless indicated otherwise,temperature is indicated in Celsius, and pressure is at or nearatmospheric.

Example 1. Humanization of an Endogenous Signal-Regulatory Protein(SIRP) Gene

This example illustrates exemplary methods of humanizing an endogenousgene encoding signal-regulatory protein alpha (SIRPα) in a non-humanmammal such as a rodent (e.g., a mouse). Human SIRPα is known to existin at least 10 allelic forms. The methods described in this example canbe employed to humanize an endogenous SIRPα gene of a non-human animalusing any human allele, or combination of human alleles (or allelefragments) as desired. In this example, human SIRPα variant 1 isemployed for humanizing an endogenous SIRPα gene of a mouse.

A targeting vector for humanization of an extracellular region of a SIRP(e.g., SIRPα) gene was constructed using VELOCIGENE® technology (see,e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659).

Briefly, mouse bacterial artificial chromosome (BAC) clone bMQ-261H14was modified to delete the sequence containing exons 2 to 4 of anendogenous SIRPα gene and insert exons 2 to 4 of a human SIRPα geneusing human BAC clone CTD-3035H21. The genomic DNA corresponding toexons 2 to 4 of an endogenous SIRPα gene (˜8555 bp) was replaced in BACclone bMQ-261H14 with a ˜8581 bp DNA fragment containing exons 2 to 4 ofa human SIRPα gene from BAC clone CTD-3035H21. Sequence analysis of thehuman SIRPα allele contained in BAC clone CTD-3035H21 revealed theallele to correspond to human variant 1. A neomycin cassette flanked byloxP sites was added to the end of the ˜8581 bp human DNA fragmentcontaining exons 2 to 4 of the human SIRPα gene (FIG. 1).

Upstream and downstream homology arms were obtained from mouse BAC DNAat positions 5′ and 3′ of exons 2 and 4, respectively, and added to the˜8581 bp human fragment-neomycin cassette to create the final targetingvector for humanization of an endogenous SIRPα gene, which containedfrom 5′ to 3′ a 5′ homology arm containing 19 kb of mouse DNA 5′ of exon2 of the endogenous SIRPα gene, a ˜8581 bp DNA fragment containing exons2 to 4 of a human SIRPα gene, a neomycin cassette flanked by loxP sites,and a 3′ homology arm containing 21 kb of mouse DNA 3′ of exon 4 of anendogenous SIRPα gene. Targeted insertion of the targeting vectorpositioned the neomycin cassette in the fifth intron of a mouse SIRPαgene between exons 4 and 5. The targeting vector was linearized bydigesting with SwaI and then used in homologous recombination inbacterial cells to achieve a targeted replacement of exons 2 to 4 in amouse SIRPα gene with exons 2 to 4 of a human SIRPα gene (FIG. 1).

The targeted BAC DNA (described above) was used to electroporate mouseES cells to created modified ES cells comprising a replacement of exons2 to 4 in an endogenous mouse SIRPα gene with a genomic fragmentcomprising exons 2 to 4 of a human SIRPα gene. Positive ES cellscontaining a genomic fragment comprising exons 2 to 4 of a human SIRPαgene were identified by quantitative PCR using TAQMAN™ probes (Lie andPetropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). The nucleotidesequence across the upstream insertion point included the following,which indicates endogenous mouse sequence upstream of the insertionpoint (contained within the parentheses below) linked contiguously to ahuman SIRPα genomic sequence present at the insertion point: (AGCTCTCCTACCACTAGACT GCTGAGACCC GCTGCTCTGC TCAGGACTCG ATTTCCAGTA CACAATCTCCCTCTTTGAAA AGTACCACAC ATCCTGGGGT) GCTCTTGCAT TTGTGTGACA CTTTGCTAGCCAGGCTCAGT CCTGGGTTCC AGGTGGGGAC TCAAACACAC TGGCACGAGT CTACATTGGATATTCTTGGT (SEQ ID NO: 6). The nucleotide sequence across the downstreaminsertion point at the 5′ end of the neomycin cassette included thefollowing, which indicates human SIRPα genomic sequence contiguous withcassette sequence downstream of the insertion point (contained withinthe parentheses below with loxP sequence italicized): GCTCCCCATTCCTCACTGGC CCAGCCCCTC TTCCCTACTC TTTCTAGCCC CTGCCTCATC TCCCTGGCTGCCATTGGGAG CCTGCCCCAC TGGAAGCCAG (TCGAG ATAACTTCGTA TAA TGTA TGCTATACGAAG TTAT ATGCATGGCC TCCGCGCCGG GTTTTGGCGC CTCCCGCGGG CGCCCCCCTCCTCACGGCGA) (SEQ ID NO: 7). The nucleotide sequence across thedownstream insertion point at the 3′ end of the neomycin cassetteincluded the following, which indicates cassette sequence contiguouswith mouse genomic sequence 3′ of exon 4 of an endogenous SIRPα gene(contained within the parentheses below): CATTCTCAGT ATTGTTTTGCCAAGTTCTAA TTCCATCAGA CCTCGACCTG CAGCCCCTAG ATAACTTCGT ATAATGTATGCTATACGAAG TTATGCTAGC (TGTCTCATAG AGGCTGGCGA TCTGGCTCAG GGACAGCCAGTACTGCAAAG AGTATCCTTG TTCATACCTT CTCCTAGTGG CCATCTCCCT GGGACAGTCA) (SEQID NO: 8). Positive ES cell clones were then used to implant female miceusing the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 andPoueymirou et al. 2007, F0 generation mice that are essentially fullyderived from the donor gene-targeted ES cells allowing immediatephenotypic analyses Nature Biotech. 25(1):91-99) to generate a litter ofpups containing an insertion of exons 2 to 4 of a human SIRPα gene intoan endogenous SIRPα gene of a mouse.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(supra). Mice bearing the humanization of exons 2 to 4 of an endogenousSIRPα gene were identified by genotyping using a modification of alleleassay (Valenzuela et al., supra) that detected the presence of the humanSIRPα gene sequences.

Mice bearing the humanized SIRPα gene construct (i.e., containing humanSIRPα exons 2 to 4 in a mouse SIRPα gene) can be bred to a Cre deletormouse strain (see, e.g., International Patent Application PublicationNo. WO 2009/114400) in order to remove any loxed neomycin cassetteintroduced by the targeting vector that is not removed, e.g., at the EScell stage or in the embryo. Optionally, the neomycin cassette isretained in the mice.

Pups are genotyped and a pup heterozygous for the humanized SIRPα geneconstruct is selected for characterization.

Example 2. Expression of Humanized SIRPα in Non-Human Animals

This example illustrates the characteristic expression of SIRPα proteinon the surface of cells from non-human animals engineered to contain anhumanized SIRPα gene construct as described in Example 1 at anendogenous SIRPα locus.

Briefly, spleens were isolated from wild type (WT) and mice heterozygousfor a humanized SIRPα gene. Spleens were then perfused with CollagenaseD (Roche Bioscience) and erythrocytes were lysed with ACK lysis bufferaccording to manufacturer's specifications. Cell surface expression ofmouse and human SIRPα was analyzed by FACS using fluorochrome-conjugatedanti-CD3 (17A2), anti-CD19 (1D3), anti-CD1 b (M1/70), anti-human SIRPα(SE5A5), and anti-mouse SIRPα (P84). Flow cytometry was performed usingBD LSRFORTESSA™. Exemplary expression of human and mouse SIRPα asdetected on the surface of CD11b+ monocytes is shown in FIG. 2.

As shown in FIG. 2, expression of both mouse and humanized SIRPα wereclearly detectable on the surface of CD11b⁺ monocytes from heterozygousmice.

Example 3. Human Cell Engraftment in Humanized SIRP Non-Human Animals

This example illustrates an improved engraftment of human hematopoieticstem cells in non-human animals of the present invention having ahumanized SIRPα gene.

Briefly, Rag2 KO IL2Rγ^(null) mice with or without a humanized SIRPαgene were raised under pathogen-free conditions. Newborn mice (2 to 5days old) were irradiated with 240 cGy and injected intra-hepaticallywith 1×10⁵ CD34⁺ human hematopoietic stem cells. The mice were bled 10to 12 weeks post engraftment and blood was analyzed by FACS usingfluorochrome-conjugated anti-human CD45 (HI30), anti-human CD3 (SK7),anti-human CD19 (HIB19) and anti-mouse CD45 (30-F11) to check for thereconstitution of the human immune system. The genetic background of themice is BALB/cTa×129/SvJae.

Exemplary percentages of human CD34 cells in wild type, miceheterozygous for humanized SIRPα, mice homozygous for humanized SIRPαand BALB-Rag2^(−/−)IL2Rγc^(−/−) (DKO) mice are shown in FIGS. 3-5.

As shown in this example, mice homozygous for a humanized SIRPα genedemonstrate improved engraftment of human CD34⁺ cells by providing thehighest percentage of human CD34+ cells in the periphery (e.g., blood)as compared to other strains tested.

Taken together, these data demonstrate that humanized SIRPα isfunctional in the mice as described herein through expression on thesurface of cells in the mouse and begin capable of supporting theengraftment of human CD34⁺ hematopoietic stem cells.

Example 4. Evaluating the Efficacy of Ab 1 on Raji Lymphoma Tumor Growthin BRG Mice

Summary

Ab 1 is bispecific antibody (bsAb) that binds to CD3, a T cell antigenassociated with the T cell receptor (TCR) complex, and CD20, a B cellsurface antigen present on normal B cells and several B cell lineagemalignancies. Ab 1 is designed to bridge CD20-expressing cells withcytotoxic T cells by binding to the CD3 subunit of the TCR, resulting inCD20-directed polyclonal T cell killing. CD20 is a clinically validatedtarget for immunotherapy; the chimeric antibody rituximab is approvedfor treatment of Non Hodgkin Lymphomas (NHL) and Chronic LymphocyticLeukemia (CLL). Although patients may become refractory to rituximab,loss of expression of CD20 is not typically observed. Therefore, abispecific antibody bridging CD20-positive tumor cells with cytotoxic Tcells represents a potential anti-tumor strategy.

In this study, the effect of treatment with CD20×CD3 bsAb Ab 1 on humanB cell lymphoma (Raji) tumor growth was examined in a mouse tumor model.The model utilized hCD34+ engrafted BALB/c-Rag2null IL2rγnull (BRG) micethat were humanized for SIRPα. These mice, with human T, B, and NKcells, as well as granulocytes, monocytes, and dendritic cells (DCs),were treated with Ab 1 twice weekly, resulting in significantsuppression of Raji tumor growth compared to vehicle control and thenon-binding control mAb, Control Ab 5. Ab 1 treatment suppressed tumorgrowth at both 0.4 mg/kg and 0.04 mg/kg with greater significance thanthe vehicle control group throughout the treatment period (p<0.0001). Nosignificant weight loss was observed in any treatment group. Theseresults show that Ab 1 targets Raji tumors in mice with human immunecells, resulting in significant tumor suppression.

Materials and Methods

Materials

Test Compound and Control Antibody

Test compound: Ab 1.

Control antibody: Control Ab 5.

Reagents

TABLE 4 Reagent List Reagent Source Identification Raji cells Regeneroncore Raji P 1-4-10 Passage #4 facility Human CD34+ Advancedhematopoietic stem Biosciences cells (HSC) isolated Resource, Inc. fromhuman fetal livers hPBMCs Reachbio Catalog #0500-300, Lot #130322L-Histidine Amresco Catalog #181164-100G, Lot #3363E344 SucroseBiosolutions Catalog #BIO640-07, Lot #0816012 RPMI Irvine ScientificCatalog #9160, Lot #9160100803 FBS Tissue Culture Catalog #101, Lot#107062 Biologicals Penicillin/ Gibco Catalog #10376-016, LotStreptomycin/ #1411480 L-Glutamine 2-Mercaptoethanol Gibco Catalog#21985-023, Lot #762405 Anti-human CD45 Invitrogen Catalog #MHCD4518,Clone H130 Anti-human NKp46 BD Biosciences Catalog #558051, Clone 9E2Anti-human CD19 BD Biosciences Catalog #555412, Clone HIB19 Anti-humanCD3 Invitrogen Catalog #MHCD0328, Clone S4.1 Anti-human CD14 BDBiosciences Catalog #557742, Clone M5E2 Anti-human CD45 BD BiosciencesCatalog #557659, Clone 30-F11 BD Fortessa BD Biosciences Special OrderInstrumentTest Systems

The tumor studies presented in this report employed 24-32 week old maleand female BALB/c-Rag2null IL2rγnull (BRG) immunodeficient micehumanized for the signal regulatory protein alpha (SIRPα) gene. Thesewere generated at Regeneron by embryonic stem (ES) cell targeting(Strowig et al., Proc Natl Acad Sci USA, 108(32): 13218-13223 (2011)).Upon recognition of CD47, SIRPα inhibits clearance of CD47 positivecells by macrophages. Previous studies have shown that BRG miceexpressing the human SIRPα transgene have enhanced engraftment of humanHSC (Strowig et al., Proc Natl Acad Sci USA, 108(32): 13218-13223(2011)).

Newborn SIRPα BRG pups were irradiated and engrafted with hCD34+hematopoietic progenitor cells derived from fetal liver (Traggiai, etal., Science, 304(5667): 104-107 (2004)). These human HSCs give rise tohuman T, B, and NK cells, as well as granulocytes, monocytes, anddendritic cells (DCs). Due to the low levels of circulating human Bcells, there are low levels of circulating human IgG. Furthermore, thesemice do not develop germinal centers, lack lymph nodes and have limitedT and B cell replenishment if these cells are depleted. Murinemonocytes, DCs, and granulocytes remain present as well. Immune cellcomposition was confirmed by flow cytometry of blood, and mice wererandomized by % human CD45 engraftment prior to use in tumor studies.Mice were implanted with Raji tumor cells at Day 0, and the ability ofAb 1 to block tumor growth over 4 weeks was tested. Body weights andtumor volumes were recorded on days 3, 6, 9, 13, 16, 20, 23, 27, 30 and34 following implantation.

Experimental Design

Reconstitution of Human Immune System in SIRPα BRG Mice

Immunodeficient BALB/c Rag2/−γc−/−(BRG) human SIRP alpha (SIRPα BRG)mice were bred in the germ-free isolators in the Regeneron animalfacility. Neonate mice were irradiated with one dose of 300cGrey, 8-24 hprior to injection of human CD34+ hematopoietic stem cells (HSC)isolated from human fetal livers. The engraftment was allowed to developfor 12-16 weeks and the number of engrafted cells was periodicallyevaluated by flow cytometry. For the entire duration of the experiment,animals were housed in the Regeneron animal facility under standardconditions in a 12-hour day/night rhythm with access to food and waterad libitum. The number of animals per cage was limited to a maximum of 5mice.

Mouse blood was analyzed to determine percent engraftment levels priorto initiating the study. Whole blood was collected into two capillarytubes containing 150 uL of 2% EDTA (ethylenediaminetetraacetic acid; 15mg/mL). Red blood cells were lysed using ACK lysing buffer for 3 minutesand the buffer was neutralized with PBS (no calcium or magnesium). Cellswere blocked with Fc Block for 5 minutes at 4° C. and then stained withhuman CD45, NKp46, CD19, CD3 and CD14 for 30 minutes at 4° C. Sampleswere analyzed by 5-laser flow cytometry (BD Fortessa). Percentengraftment was determined as the % human CD45+ cells of total cells.

Raji Tumor Study Procedure in SIRPα BRG Mice

On day 0, groups of 5 SIRPα BRG mice were administered 2×10⁶ Raji tumorcells subcutaneously. On the same day, mice were treated with anintraperitoneal (IP) dose of either Ab 1 (0.4 or 0.04 mg/kg),non-binding control mAb Control Ab 5 (which binds a feline antigen withno cross-reactivity to human CD20 or CD3) at a dose of 0.4 mg/kg orvehicle alone. Mice subsequently received two doses of antibody/week for4 weeks. Tumor growth was measured with calipers on days 3, 6, 9, 13,16, 20, 23, 27, 30 and 34. Study groups are summarized in Table 5.

TABLE 5 Summary of Treatment Groups in SIRPα BRG Mice Dose # GroupsTumor Antibody (mg/kg) Route Schedule Mice Control Raji No antibody 0 IP2x/wk 5 Groups (Vehicle alone) Raji Control Ab 5 0.4 IP 2x/wk 5Experimental Raji Ab 1 0.4 IP 2x/wk 5 Groups Raji Ab 1 0.04 IP 2x/wk 5Specific ProceduresPreparation of Reagents

Ab 1 and Control Ab 5 were each diluted to the desired concentration inVehicle (10 mM histidine, 5% sucrose, pH 5.8). Raji cells were obtainedfrom the Regeneron core facility (passage 4) and maintained in culturemedia: RPMI 1640+10% FBS+Pen Strep-L-Glu+Mercaptoethanol. Raji cellswere diluted to the desired concentration in media.

Statistical Analyses

Statistical analyses were performed utilizing GraphPad software Prism5.0 (MacIntosh Version). Statistical significance was determined bytwo-way ANOVA with Tukey's multiple comparisons post-test. Data fromeach of the readouts were compared across treatment groups. A thresholdof p<0.05 was considered statistically significant, as indicated by *.Mice that died prior to the end of study were removed from the combinedtumor growth curve (but not the individual mouse growth curve) graphs asindicated and statistical analysis in order to analyze by two-way ANOVA.

Results

Ab 1 Suppresses Raji Tumor Cell Growth in hCD34+ Engrafted SIRPα BRGMice

Ab 1 suppressed Raji tumor growth compared to vehicle control andnon-binding control in hCD34+ engrafted SIRPα BRG mice (FIG. 6). NewbornSIRPα BRG pups were irradiated and engrafted with hCD34+ fetal livercells as hematopoietic progenitor cells (Traggiai, et al., Science,304(5667): 104-107 (2004)), which gave rise to human T, B, and NK cells,as well as granulocytes, monocytes, and DCs. On day 0, hCD34+ engraftedSIRPα BRG mice were administered 2×10⁶ Raji tumor cells subcutaneously.On the same day, mice were treated with an intraperitoneal (IP) dose ofeither Ab 1 (0.4 or 0.04 mg/kg) or the non-binding control mAb ControlAb 5, or vehicle control, followed by twice weekly doses throughout thestudy.

Compared to the vehicle control groups and the non-binding controlgroups, Ab 1 significantly suppressed Raji tumor outgrowth administeredat doses of 0.04 mg/kg (p<0.0001) or 0.4 mg/kg (p<0.0001) on day 34 posttumor implantation (FIG. 7). Furthermore, the effects of Ab 1 treatmentwere dose-dependent, with 0.4 mg/kg Ab 1 suppressing growth completelythroughout the study, as compared to 0.04 mg/kg Ab 1, which suppressedtumor growth completely by Day 30. Neither Ab 1 nor the non-bindingcontrol mAb had a significant effect on mouse body weight throughout thestudy (FIG. 8).

CONCLUSION

The effect of treatment with Abl, a CD20×CD3 bsAb, on Raji tumor growthwas examined in a mouse model. Ab 1 was effective in tumor growthsuppression in hCD34+ engrafted SIRPα BRG mice with human T, B, and NKcells, as well as granulocytes, monocytes, and DCs. Twice weeklytreatment with Ab 1 resulted in significant and dose-dependentsuppression of Raji human B cell lymphoma tumor growth compared tovehicle control and non-binding control. No significant weight loss wasobserved in any treatment group. These results show that Ab 1 targetsRaji tumors in mice with human immune cells, resulting in significanttumor growth suppression.

EQUIVALENTS

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated by those skilled in the art thatvarious alterations, modifications, and improvements will readily occurto those skilled in the art. Such alterations, modifications, andimprovements are intended to be part of this disclosure, and areintended to be within the spirit and scope of the invention.Accordingly, the foregoing description and drawing are by way of exampleonly and the invention is described in detail by the claims that follow.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification.

Those skilled in the art will appreciate typical standards of deviationor error attributable to values obtained in assays or other processesdescribed herein.

The publications, websites and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

We claim:
 1. A rodent whose genome comprises a humanized SIRPα gene atan endogenous rodent SIRPα locus, wherein the humanized SIRPα gene (i)comprises exon 1 of a rodent SIRPα gene, exons 2, 3 and 4 of a humanSIRPα gene, and exons 5, 6, 7 and 8 of the rodent SIRPα gene, (ii) isoperably linked to a rodent SIRPα promoter at the endogenous rodentSIRPα locus, and (iii) expresses in the rodent a humanized SIRPα proteincomprising an extracellular portion of the human SIRPα protein encodedby the human SIRPα gene and an intracellular portion of the rodent SIRPαprotein encoded by the rodent SIRPα gene; and wherein the rodent is arat and is homozygous for the humanized SIRPα gene.
 2. The rodent ofclaim 1, wherein the extracellular portion of the human SIRPα proteincomprises amino acid residues 28-362 of the human SIRPα protein.
 3. Therodent of claim 1, wherein the human SIRPα protein comprises the aminoacid sequence as set forth in SEQ ID NO:
 4. 4. The rodent of claim 1,wherein the rodent does not express an endogenous rodent SIRPα protein.5. A rodent embryonic stem (ES) cell, comprising a humanized SIRPα geneat an endogenous rodent SIRPα locus, wherein the humanized SIRPα gene(i) comprises exon 1 of a rodent SIRPα gene, exons 2, 3 and 4 of a humanSIRPα gene, and exons 5, 6, 7 and 8 of the rodent SIRPα gene, (ii) isoperably linked to a rodent SIRPα promoter at the endogenous rodentSIRPα locus, and (iii) encodes a humanized SIRPα protein comprising anextracellular portion of the human SIRPα protein encoded by the humanSIRPα gene and an intracellular portion of the rodent SIRPα proteinencoded by the rodent SIRPα gene; and wherein the rodent ES cell is arat ES cell.
 6. The rodent ES cell of claim 5, wherein the extracellularportion of the human SIRPα protein comprises amino acid residues 28-362of the human SIRPα protein.
 7. The rodent ES cell of claim 5, whereinthe human SIRPα protein comprises the amino acid sequence as set forthin SEQ ID NO:
 4. 8. The rodent ES cell of claim 5, wherein the rodent EScell is homozygous for the humanized SIRPα gene.
 9. A rodent embryocomprising the rodent ES cell of claim 5, wherein the rodent embryo is arat embryo.